Methods of combined bioprocessing and related microorganisms, thermophilic and/or acidophilic enzymes, and nucleic acids encoding said enzymes

ABSTRACT

A genetically modified organism comprising: at least one nucleic acid sequence and/or at least one recombinant nucleic acid isolated from  Alicyclobacillus acidocaldarius  and encoding a polypeptide involved in at least partially degrading, cleaving, transporting, metabolizing, or removing polysaccharides, cellulose, lignocellulose, hemicellulose, lignin, starch, sugars, sugar oligomers, carbohydrates, complex carbohydrates, chitin, heteroxylans, glycosides, xylan-, glucan-, galactan-, or mannan-decorating groups; and at least one nucleic acid sequence and/or at least one recombinant nucleic acid encoding a polypeptide involved in fermenting sugar molecules to a product. Additionally, enzymatic and/or proteinaceous extracts may be isolated from one or more genetically modified organisms. The extracts are utilized to convert biomass into a product. Further provided are methods of converting biomass into products comprising: placing the genetically modified organism and/or enzymatic extracts thereof in fluid contact with polysaccharides, cellulose, lignocellulose, hemicellulose, lignin, starch, sugars, sugar oligomers, carbohydrates, complex carbohydrates, chitin, heteroxylans, glycosides, and/or xylan-, glucan-, galactan-, or mannan-decorating groups.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.15/676,484, filed Aug. 14, 2017, pending, which is a continuation ofU.S. patent application Ser. No. 15/074,297, filed Mar. 18, 2016, nowU.S. Pat. No. 9,732,330, which is a continuation-in-part of U.S. patentapplication Ser. No. 14/185,501, filed Feb. 20, 2014, now U.S. Pat. No.9,290,784, issued Mar. 22, 2016, which is a continuation-in-part of U.S.patent application Ser. No. 13/924,149, filed Jun. 21, 2013, now U.S.Pat. No. 8,691,525, issued Apr. 14, 2014, which is a divisional of U.S.patent application Ser. No. 12/587,229, filed Oct. 2, 2009, now U.S.Pat. No. 8,492,114, issued Jul. 23, 2013, which is acontinuation-in-part of U.S. patent application Ser. No. 12/322,359,filed Jan. 29, 2009, now U.S. Pat. No. 7,858,353, issued Dec. 28, 2010,to Thompson et al., which claims the benefit of U.S. Provisional PatentApplication Ser. No. 61/025,136, filed Jan. 31, 2008, for “THERMOPHILICAND THERMOACIDOPHILIC BIOPOLYMER-DEGRADING GENES AND ENZYMES FROMALICYCLOBACILLUS ACIDOCALDARIUS AND RELATED ORGANISMS, METHODS”; andU.S. patent application Ser. No. 12/587,229 is a continuation-in-part ofU.S. patent application Ser. No. 12/380,551, filed Feb. 26, 2009, nowU.S. Pat. No. 8,728,803, issued May 20, 2014, to Thompson et al., whichclaims the benefit of U.S. Provisional Patent Application Ser. No.61/032,339, filed Feb. 28, 2008, for “THERMOPHILIC AND THERMOACIDOPHILICMETABOLISM GENES AND ENZYMES FROM ALICYCLOBACILLUS ACIDOCALDARIUS ANDRELATED ORGANISMS, METHODS”; and U.S. patent application Ser. No.12/587,229 is a continuation-in-part of U.S. patent application Ser. No.12/380,450, filed Feb. 26, 2009, now U.S. Pat. No. 9,234,228, issuedJan. 12, 2016, to Thompson et al., which claims the benefit of U.S.Provisional Patent Application Ser. No. 61/031,984, filed Feb. 27, 2008,for “THERMOPHILIC AND THERMOACIDOPHILIC GLYCOSYLATION GENES AND ENZYMESFROM ALICYCLOBACILLUS ACIDOCALDARIUS AND RELATED ORGANISMS, METHODS”;and U.S. patent application Ser. No. 12/587,229 is acontinuation-in-part of U.S. patent application Ser. No. 12/380,554,filed Feb. 26, 2009, now U.S. Pat. No. 7,960,534, issued Jun. 14, 2011,to Thompson et al., which claims the benefit of U.S. Provisional PatentApplication Ser. No. 61/031,593, filed Feb. 26, 2008, for “THERMOPHILICAND THERMOACIDOPHILIC SUGAR TRANSPORTING GENES AND ENZYMES FROMALICYCLOBACILLUS ACIDOCALDARIUS AND RELATED ORGANISMS, METHODS”; andU.S. patent application Ser. No. 12/587,229 is a continuation-in-part ofU.S. patent application Ser. No. 12/380,008, filed Feb. 20, 2009, nowU.S. Pat. No. 8,716,011, issued May 6, 2014, to Lee et al., which claimsthe benefit of U.S. Provisional Patent Application Ser. No. 61/030,820,filed Feb. 22, 2008, for “TRANSCRIPTIONAL CONTROL IN ALICYCLOBACILLUSACIDOCALDARIUS AND ASSOCIATED GENES, PROTEINS, AND METHODS”; and U.S.patent application Ser. No. 12/587,229 is a continuation-in-part of U.S.patent application Ser. No. 12/321,636, filed Jan. 23, 2009, now U.S.Pat. No. 7,923,234, issued Apr. 12, 2011, to Thompson et al., whichclaims the benefit of U.S. Provisional Patent Application Ser. No.61/023,639, filed Jan. 25, 2008, for “THERMAL AND ACID TOLERANTBETA-XYLOSIDASES, GENES ENCODING, RELATED ORGANISMS, AND METHODS”; thedisclosure of each of which is hereby incorporated herein in itsentirety by this reference.

This application is related to U.S. patent application Ser. No.14/887,073, filed Oct. 19, 2015, pending, which is a divisional of U.S.patent application Ser. No. 14/226,573, filed Mar. 26, 2014, now U.S.Pat. No. 9,222,094, issued Dec. 29, 2015, which is a divisional of U.S.patent application Ser. No. 12/380,551, filed Feb. 26, 2009, now U.S.Pat. No. 8,728,803, issued May 20, 2014, to Thompson et al. which claimsthe benefit of U.S. Provisional Patent Application Ser. No. 61/025,136,filed Jan. 31, 2008. This application is also related to U.S. patentapplication Ser. No. 14/977,349, filed Dec. 21, 2015, pending, which isa divisional of U.S. patent application Ser. No. 12/380,450, filed Feb.26, 2009, now U.S. Pat. No. 9,234,228, issued Jan. 12, 2016, to Thompsonet al., which claims the benefit of U.S. Provisional Patent ApplicationSer. No. 61/031,984, filed Feb. 27, 2008. This application is alsorelated to U.S. patent application Ser. No. 13/721,172, filed Dec. 20,2012, now U.S. Pat. No. 8,575,323, issued Nov. 5, 2013, which is adivisional of U.S. patent application Ser. No. 13/517,887, filed Jun.14, 2012, now U.S. Pat. No. 8,362,226, issued Jan. 29, 2013, which is adivisional of U.S. patent application Ser. No. 13/200,164, filed Sep.20, 2011, now U.S. Pat. No. 8,354,517, issued Jan. 15, 2013 which is adivisional of U.S. patent application Ser. No. 13/066,645, filed Apr.19, 2011, now U.S. Pat. No. 8,071,748, issued Dec. 6, 2011, which is adivisional of U.S. patent application Ser. No. 12/380,554, filed Feb.26, 2009, now U.S. Pat. No. 7,960,534, issued Jun. 14, 2011, to Thompsonet al., which claims the benefit of U.S. Provisional Patent ApplicationSer. No. 61/031,593, filed Feb. 26, 2008. This application is alsorelated to U.S. patent application Ser. No. 14/920,598, filed Oct. 22,2015, now U.S. Pat. No. 9,499,824, issued Nov. 22, 2016, which is adivisional of U.S. patent application Ser. No. 14/256,819, filed Apr.18, 2014, now U.S. Pat. No. 9,187,753, issued Nov. 17, 2015 which is adivisional of U.S. patent application Ser. No. 12/380,008, filed Feb.20, 2009, now U.S. Pat. No. 8,716,011, issued May 6, 2014, to Lee etal., which claims the benefit of U.S. Provisional Patent ApplicationSer. No. 61/030,820, filed Feb. 22, 2008, the disclosure of each ofwhich is hereby incorporated herein in its entirety by this reference.

GOVERNMENT RIGHTS

This invention was made with government support under Contract NumberDE-AC07-99ID13727 and Contract Number DE-AC07-05ID14517 awarded by theUnited States Department of Energy. The government has certain rights inthe invention.

STATEMENT ACCORDING TO 37 C.F.R. § 1.821(c) or (e)—SEQUENCE LISTINGSUBMITTED AS A TXT

Pursuant to 37 C.F.R. § 1.821(c) or (e), files containing a TXT versionof the Sequence Listing have been submitted concomitant with thisapplication, the contents of which are hereby incorporated by reference.

BACKGROUND

Dilute acid hydrolysis to remove hemicellulose from lignocellulosicmaterials is one of the most developed pretreatment techniques forlignocellulose and is currently favored (Hamelinck et al., 2005) becauseit results in fairly high yields of xylose (75% to 90%). Conditions thatare typically used range from 0.1 to 1.5% sulfuric acid and temperaturesabove 160° C. The high temperatures used result in significant levels ofthermal decomposition products that inhibit subsequent microbialfermentations (Lavarack et al., 2002). High temperature hydrolysisrequires pressurized systems, steam generation, and corrosion resistantmaterials in reactor construction due to the more corrosive nature ofacid at elevated temperatures.

Lower temperature acid hydrolyses are of interest because they have thepotential to overcome several of the above shortcomings (Tsao et al.,1987). It has been demonstrated that 90% of hemicellulose can besolubilized as oligomers in a few hours of acid treatment in thetemperature range of 80° C. to 100° C. It has also been demonstratedthat the sugars produced in low temperature acid hydrolysis are stableunder those same conditions for at least 24 hours with no detectabledegradation to furfural decomposition products. Finally, sulfuric acidtypically used in pretreatments is not as corrosive at lowertemperatures. The use of lower temperature acid pretreatments requiresmuch longer reaction times to achieve acceptable levels of hydrolysis.Although 90% hemicellulose solubilization has been shown (Tsao, 1987),the bulk of the sugars are in the form of oligomers and are not in themonomeric form. The organisms currently favored in subsequentfermentation steps cannot utilize sugar oligomers (Garrote et al., 2001)and the oligomer-containing hydrolysates require further processing tomonomers, usually as a second acid or alkaline hydrolysis step (Garroteet al., 2001).

Other acidic pretreatment methods include autohydrolysis and hot waterwashing. In autohydrolysis, biomass is treated with steam at hightemperatures (˜240° C.), which cleaves acetyl side chains associatedwith hemicellulose to produce acetic acid that functions in a similarmanner to sulfuric acid in acid hydrolysis. Higher pretreatmenttemperatures are required as compared to dilute sulfuric acid hydrolysisbecause acetic acid is a much weaker acid than sulfuric. At temperaturesbelow 240° C., the hemicellulose is not completely hydrolyzed to sugarmonomers and has high levels of oligomers (Garrote et al., 2001). In hotwater washing, biomass is contacted with water (under pressure) atelevated temperatures 160° C. to 220° C. This process can effectivelyhydrolyze greater than 90% of the hemicellulose present, and thesolubilized hemicellulose was typically over 95% in the form ofoligomers (Liu and Wyman, 2003).

BRIEF SUMMARY

The entire contents of each of the following applications and patentsare incorporated herein in their entirety by this reference: patentapplication Ser. No. 12/322,359, filed Jan. 29, 2009, now U.S. Pat. No.7,858,353, issued Dec. 28, 2010, to Thompson et al.; Ser. No.12/321,636, filed Jan. 23, 2009, now U.S. Pat. No. 7,923,234, issuedApr. 12, 2011, to Thompson et al.; Ser. No. 12/380,008 filed Feb. 20,2009, now U.S. Pat. No. 8,716,011, issued May 6, 2014, to Lee et al.;Ser. No. 12/380,554, filed Feb. 26, 2009, now U.S. Pat. No. 7,960,534,issued Jun. 14, 2011, to Thompson et al.; Ser. No. 12/380,450 (filedFeb. 26, 2009); and Ser. No. 12/380,551 filed Feb. 26, 2009, now U.S.Pat. No. 8,728,803, issued May 20, 2014, to Thompson et al.

Embodiments of the invention relate to a genetically modified organismfor converting biomass into products. The genetically modified organismmay comprise at least one nucleic acid sequence encoding a polypeptideas disclosed in the patent applications and patents previouslyincorporated by reference herein. In embodiments, the geneticallymodified organism may comprise at least one nucleic acid sequenceencoding a polypeptide associated with at least partially degrading,cleaving, transporting, metabolizing and/or removing polysaccharides,cellulose, hemicellulose, lignin, starch, sugars, sugar oligomers,carbohydrates, complex carbohydrates, chitin, heteroxylans, glycosides,xylan-, glucan-, galactan-, or mannan-decorating groups; and/or at leastone nucleic acid sequence encoding a polypeptide associated withfermenting sugar molecules to products.

Embodiments of the invention also relate to protein(s) and/or cellularextracts isolated from a genetically modified organism. The isolatedprotein and/or cellular extracts may comprise: at least one polypeptideisolated from a genetically modified organism, the organism including:at least one recombinant nucleic acid encoding a polypeptide asdisclosed in the patent applications and patents previously incorporatedby reference herein. In embodiments, the at least one recombinantnucleic acid encoding a polypeptide may comprise: at least onepolypeptide involved in at least partially degrading, cleaving,transporting, metabolizing and/or removing polysaccharides, cellulose,hemicellulose, lignin, starch, sugars, sugar oligomers, carbohydrates,complex carbohydrates, chitin, heteroxylans, glycosides, xylan-,glucan-, galactan-, or mannan-decorating groups; and/or at least onerecombinant nucleic acid encoding a polypeptide involved in fermentingsugar molecules to products.

Additional embodiments of the invention relate to methods of at leastpartially processing polysaccharides, cellulose, hemicellulose, starch,sugars, sugar oligomers, carbohydrates, complex carbohydrates, chitin,heteroxylans, glycosides, xylan-, glucan-, galactan-, ormannan-decorating groups into a product. The method may comprise:placing a genetically modified organism in fluid contact with apolysaccharide, cellulose, hemicellulose, starch, sugars, sugaroligomers, carbohydrates, complex carbohydrates, chitin, heteroxylans,glycoside, xylan-, glucan-, galactan-, and/or mannan-decorating group.The genetically modified organism may comprise at least one nucleic acidand/or at least one recombinant nucleic acid encoding a polypeptideinvolved in at least partially degrading, cleaving, transporting,metabolizing, or removing polysaccharides, cellulose, hemicellulose,lignin, starch, sugars, sugar oligomers, carbohydrates, complexcarbohydrates, chitin, heteroxylans, glycosides, xylan-, glucan-,galactan-, or mannan-decorating groups; and/or at least one nucleic acidand/or at least one recombinant nucleic acid encoding a functionalprotein involved in fermenting sugar molecules to a product and/or afermentation enzyme involved in converting sugars into a product.

Further embodiments of the invention relate to isolating an extract froma genetically modified organism according to the present invention.Embodiments may also include placing the isolated extract in fluidcontact with a polysaccharide, cellulose, hemicellulose, lignin, starch,sugars, sugar oligomers, carbohydrates, complex carbohydrates, chitin,heteroxylans, glycoside, xylan-, glucan-, galactan-, and/ormannan-decorating group. The genetically modified organism may compriseat least one nucleic acid and/or at least one recombinant nucleic acidencoding a polypeptide having some level of activity in at leastpartially degrading, cleaving, transporting, metabolizing, and/orremoving polysaccharides, cellulose, hemicellulose, lignin, starch,sugars, sugar oligomers, carbohydrates, complex carbohydrates, chitin,heteroxylans, glycosides, xylan-, glucan-, galactan-, ormannan-decorating groups; and/or at least one nucleic acid and/or atleast one recombinant nucleic acid encoding a functional proteininvolved in fermenting sugar molecules to a product such as afermentation or other enzyme involved in converting sugars into aproduct.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-ID depict a sequence alignment between SEQ ID NO:337(RAAC02661), a xylan alpha-1,2-glucuronidase, and gi|15613624,gi|118725970, gi|148270004, gi|5642830, and gi|16621784 (SEQ IDNOs:338-342, respectively) which are all xylan alpha-1,2-glucuronidases.Amino acids common to three or more of the sequences aligned areindicated in bold.

FIG. 2 is a graphical representation of the relative α-glucuronidase(AGUR) activity of RAAC02661 (SEQ ID NO:337) produced in E. coli.Diamonds indicate the activity at 50° C., squares indicate the activityat 60° C., triangles indicate the activity at 70° C., Xs indicate theactivity at 80° C., and circles indicate the activity at 90° C.

DETAILED DESCRIPTION

Lignocellulose is a highly heterogeneous three-dimensional matrixcomprised of cellulose, hemicellulose, and lignin. Many fuels andchemicals can be made from these lignocellulosic materials. To utilizelignocellulosic biomass for production of fuels and chemicals viafermentative processes, it is necessary to convert the plantpolysaccharides to simpler sugars, which are then fermented to productsusing a variety of organisms. Direct hydrolysis of cellulose by mineralacids to monomers is possible at high temperature and pressure, leadingto yield losses due to thermal decomposition of the sugars. One strategyto reduce these yield losses is to use cellulases and potentially otherenzymes to depolymerize the polysaccharides at moderate temperatures.Addition of acid-stable thermotolerant hydrolytic enzymes such ascellulases and xylanases to the biomass slurry during the pretreatmentallows the use of lower temperatures and pressures, as well as cheapermaterials of reactor construction, reducing both the capital and energycosts. Another approach is to combine the reduced severity pretreatmentwith enzymes together with fermentation under the same conditions, usinga single organism that produces the enzymes to degrade the material aswell as ferment the sugars to the value-added product of choice.

For commercially available enzymes to be used for this purpose, thepretreatment slurry must be neutralized and cooled to 40° C. to 50° C.,adding significant cost to the process. Hence, it would be animprovement in the art to degrade the soluble oligomers produced usingacid, autohydrolysis or hot water washing pretreatments, at reducedseverity and without neutralization using, for example, thermophilicand/or acidophilic enzymes.

Embodiments of the invention relate to a genetically modified organismcomprising at least one of a nucleic acid sequence and protein sequenceencoded by a nucleic acid sequence from Alicyclobacillus acidocaldarius.Such nucleic acids may include any of those nucleotide sequencesdescribed in the patent applications and patents incorporated herein byreference. Such nucleic acids include, but are not limited to, thosecoding for enzymes capable of depolymerizing cellulosic polysaccharidesto simpler carbohydrates and their transport into the bacterial cell andmetabolism within the cell. Enzyme activities associated with transportinto the cell and metabolism within the cell may be thermophilic and/oracidophilic in nature and general examples of nucleic acids coding forsimilar enzymes are described in the literature. Enzyme activitiesassociated with cellulose depolymerization and/or metabolism may bethermophilic (intracellular) and/or thermoacidophilic (extracellular)and include, without limitation, the following classes of enzymes:Alpha-glucosidases, Glucan 1,4-alpha-maltohydrolases, Glycosidases,Amylases, Acetyl esterases, Beta-galactosidases, Alpha amylases,Alpha-xylosidases, Cyclomaltodextrinases; Neopullulanases; Maltogenicalpha-amylases, Family 31 of glycosyl hydrolase,Alpha-L-arabinofuranosidases, Altronate hydrolases,poly-1,4-alpha-D-galacturonidase, Xylan alpha-1,2-glucuronosidases,Cellulase/Endoglucanase, Polygalacturonases, Glycosyl hydrolases,Peptidoglycan hydrolases, N-acetylglucosaminidases, Endochitinases,Alpha-galactosidases, Endo-beta-1,4-mannanases, Cellobiosephosphorylases, Cyclic beta-1,2-glucan synthases, Glycogen debranchingenzymes, Acetyl hydrolases, Beta-1,4-xylanases, Beta-glucosidases,6-phospho-beta-glucosidases, Cinnamoyl ester hydrolases,Beta-glucuronidases, Xylan alpha-1,2-glucuronosidases,3-hydroxyisobutyryl-CoA hydrolases, glycosidases, Chitooligosaccharidedeacetylases, glycosyl hydrolases (or glycoside hydrolases); esterasesincluding acetylxylan esterases and p-cumaric acid esterases and ferulicacid esterases; and/or uronidases.

Additionally, embodiments of the invention relate in part to agenetically modified organism comprising, in addition to at least one ofthe nucleic acid sequences and protein sequences encoded by nucleicacids of Alicyclobacillus acidocaldarius, at least one of the nucleicacid sequences and protein sequences associated with fermenting sugarmolecules to products. Such nucleic acid sequences may code for proteinsequences which may be thermophilic and/or acidophilic in nature andgeneral examples of similar nucleic acids are described in theliterature.

The present invention also relates to isolated and/or purifiednucleotide sequences from the genome of Alicyclobacillus acidocaldariusselected from the sequences described in the patent applications andpatents previously incorporated herein by reference.

Nucleotide, polynucleotide, or nucleic acid sequence will be understoodaccording to the present invention as meaning either double-stranded orsingle-stranded DNA and the transcription products of said DNAs.

As used herein, “fermentation” relates to the biological conversion of asugar molecule into a product. As used herein, a “product” is anychemical that can be made, at least in part, through a biologicalprocess. Examples of products include, but are not limited to, ethanol,acetic acid, glyoxylic acid, oxalic acid, lactic acid,3-hydroxypropionic acid, glycerol, 1,2-propanediol, 1,3-propanediol,propionic acid, acetone, fumaric acid, succinic acid, malic acid,butyric acid, 1-butanol, 2,3-butanediol, acetoin, aspartic acid,1,2,-butanediol, itaconic acid, glutamic acid, citric acid, aconiticacid, cis-cis muconic acid, gluconic acid, kojic acid, amino acids,vitamins, alginate, cellulose, curdlan, chondroitin, cyanophycin,gellan, heparin, hyaluronic acid, poly-gamma-glutamic acid,poly-epsilon-lysine, polyhydroxyalkanoates, pullulan, scleroglucan,xanthan, indigo, and those chemicals set forth in the BREW report fromthe University of Utrect, (Patel, M. et al., (2006), “Medium andlong-term opportunities and risks of the biotechnological production ofbulk chemicals from renewable resources: The potential of whitebiotechnology.” The BREW Project. Final Report prepared under theEuropean Commission's GROWTH Programme (DG Research),(publica.fraunhofer.de/eprints/N-48834.pdf)), the entirety of thecontents of which are incorporated herein by this reference.

Although the following sections related to Hydrolysis AssociatedMolecules (HAMs) and Fermentation Associated Molecules (FAMs), thetechniques therein apply equally to all other nucleotide sequencesisolated and/or purified from the genome of acidophilic and orthermophilic organisms, such as, without limitation, Alicyclobacillusacidocaldarius and those HAMs and FAMs described in the patentapplications and patents previously incorporated herein by reference.

Expression/Integration of Hydrolysis Associated Molecules

In embodiments of the invention, one or more Hydrolysis AssociatedMolecules (HAMs) may be incorporated and/or inserted into an organismthat has the ability to ferment sugars to products. The HAMs may includeregulatory factors and/or nucleic acids coding for proteins associatedwith, involved in, and/or assisting in the breakdown and/or hydrolysisof biomass (i.e., polysaccharides, cellulose, lignocellulose,hemicellulose, lignin, starch, sugars, sugar oligomers, carbohydrates,complex carbohydrates, chitin, heteroxylans, glycosides, xylan-,glucan-, galactan-, or mannan-decorating groups, etc.) into more simplesugar molecules and/or sugar monomers. A non-exhaustive list of theseproteins and/or enzymes include: cellulases (i.e., endo- and/orexo-cellulases such as endo-beta-1,4-glucanase orexo-beta-1,4-glucanase); hemicellulases (i.e., exo- and/orendo-beta-1,4-xylanase); one or more beta-xylosidase enzymes;β-1,4-cellobiohydrolases (CBH I & CBH II); xylanases (XYN I & XYN II);β-glucosidase; α-L-arabinofuranosidase; acetyl xylan esterase;β-mannanase; and α-glucuronidase; esterases of the alpha-beta hydrolasesuperfamily; alpha-beta hydrolase; alpha-glucosidases; alpha-xylosidase;alpha-L-arabinofuranosidase; altronate hydrolase; acellulose/endoglucanase; a polygalacturonase; an alpha-galactosidase; acellobiose phosphorylase; a glycogen debranching enzyme; an acetylesterase/acetyl hydrolase; a beta-1,4-xylanase; a cinnamoyl esterhydrolase; a carboxylesterase type B; abeta-galactosidase/beta-glucuronidase; a xylan alpha-1,2-glucuronidase;a 3-hydroxyisobutyryl-CoA hydrolase; a beta-glucosidase B-relatedglycosidase; and/or a chitooligosaccharide deacetylase and/or so forth.Additional proteins and/or enzymes may include ligninolytic enzymes suchas manganese peroxidase, lignin peroxidase, or the like.

Additionally, in embodiments, the HAMs may encompass all those nucleicacids and/or proteins associated with the replication, transcription,translation, and/or expression of nucleic acids involved with thebreakdown and/or hydrolysis of biomass and/or lignocellulose into moresimple sugar molecules and/or sugar monomers. In a non-limiting example,HAMs may comprise those nucleic acids involved with the regulation ofhydrolysis, such as, but not limited to, those nucleic acids and/orproteins that increase the replication, transcription, expression, etc.,of HAMs. Further, HAMs may comprise those nucleic acids and/or proteinsthat are involved with the down-regulation of other HAMs. Anon-exhaustive list of these regulatory HAMs may include: transcriptionfactors, enhancers, repressors, DNA binding proteins, or the like.

In embodiments, the HAMs may include derivatives, analogs, and/ormodified versions of one or more HAMs. These derivative, analogs, and/ormodified versions may include those modifications carried out in vivo orin vitro. Some non-limiting examples of derivative, analogs, and/ormodifications include: substitutions, deletions, mutations,modifications etc. Modifications may comprise all modificationsoccurring to nucleic acids, DNA, RNA, proteins, etc., such as, but notlimited to, acetylation, deacetylation, alkylation, methylation,demethylation, carboxylation, glycosylation, phosphorylation,hydroxylation, PEGylation, biotinylation, and/or any other type ofmodification known in the art.

In embodiments, the HAMs may encompass those nucleic acids, proteins,and/or enzymes that are derivatives of HAMs and include tags, markers,and/or other modifications. A non-exhaustive list of tags may include:His-tags, FLAG tags, Calmodulin-tags, HA-tags, Maltose bindingprotein-tags, Thioredoxin-tags, S-tags, Strep-tags, Nus-tags, or thelike. The HAMs may also include those nucleic acids, proteins, and/orenzymes that include fluorescent markers, such as, but not limited to,GFP, RFP, YFP, BFP, or the like.

In additional embodiments, HAMs may encompass nucleic acid or peptidesignals that direct secretion of HAM proteins from the cell in whichthey are produced. Examples of such signals include N-terminal and/orC-terminal sequences that direct localization and/or secretion of themolecules with which they are associated. In further embodiments, HAMsmay include transporter proteins, such as, by way of non-limitingexample, ABC transporter proteins that direct the secretion ofparticular molecules (such as other HAMs) to the extracellular orperiplasmic space. In embodiments, the signals may direct secretion of aHAM through any system for secretion including, but not limited to,Endoplasmic Reticulum/Golgi Apparatus systems; vesicle mediatedsecretion systems; any of the Type I, II, III, IV, V, VI, and/or Tatsecretion systems; and/or Sec pathway systems.

The incorporation of the HAMs into the one or more organisms with theability to ferment sugars to products may enable the one or moreorganisms to convert cellulosic and/or other biomass compositions intoproducts in a variety of pH and temperature conditions. In oneembodiment, the one or more genetically modified organisms may carry outthe biomass conversion in a range of pH conditions from at or less thana pH of about 7; at or less than a pH of about 5, about a pH of 1 toabout a pH of 5; and/or from about a pH of 1 to about a pH of 1.3.

In embodiments, the ability of a genetically modified organism accordingto the present invention to function and/or convert biomass intoproducts in pH conditions of 1 to 5, may eliminate the need toneutralize and/or increase the pH to a range of about 5 to 6.0 forenzymatic hydrolysis and/or fermentation after dilute acid pretreatment,which, using conventional methods, is necessary for the hydrolysis andfermentation of cellulose to products. Indeed, in conventional methodsof dilute acid hydrolysis, the pretreatment hydrolysate needs to beneutralized to enable enzymatic hydrolysis and fermentation to occur. Assuch, in one embodiment, the HAMs may code for thermophilic,thermoacidophilic, and/or acidophilic enzymes, properties and/orcharacteristics, or in the alternative they may be derived from one ormore thermophilic, thermoacidophilic, and/or acidophilic organisms.

It is additionally contemplated that one or more of the HAM encodedproteins may function and/or have an optimum pH range of above a pH of5. Indeed, one or more of the HAMs may function and/or enable breakdownand/or hydrolysis of biomass and/or cellulose into more simple sugarmolecules and/or sugar monomers in pH conditions that range from a pH ofabout 5 to a pH of about 14.

In embodiments, one or more HAMs may function to and/or assist in thebreakdown and/or hydrolysis of biomass and/or cellulosic materials intomore simple sugar molecules and/or sugar monomers in a broad range oftemperatures. Conventional methods and techniques of pretreatment fordilute acid hydrolysis and low temperature acid hydrolysis occur attemperature ranges of about 160 degrees Celsius and a range of eighty(80) degrees to one hundred (100) degrees Celsius, respectively.However, using the conventional methods, in order to begin the enzymatichydrolysis and fermentation reactions, the pretreatment mixture/slurryneeds to be cooled to around forty (40) degrees to fifty (50) degreesCelsius.

In contrast to the current and/or conventional methods, the HAMsincorporated into the organism may function to, assist in, and/or carryout the breakdown and/or hydrolysis of biomass and/or cellulosicmaterials into more simple sugar molecules and/or sugar monomers in abroad range of temperatures. Some non-limiting examples of temperatureranges include: at least about fifty (50) degrees Celsius; at leastabout seventy (70) degrees Celsius; from about forty-five (45) degreesCelsius to about eighty (80) degrees Celsius; from about eighty (80)degrees Celsius to about one hundred (100) degrees Celsius; from abouteighty-five (85) degrees to about ninety-five (95) degrees Celsius;and/or from about ninety (90) degrees to about one hundred (100) degreesCelsius.

In embodiments, the one or more HAMs may be isolated from, derived fromand/or originate from one or more organisms. The one or more organismsmay comprise the same organism, organisms within the same genus and/orwithin the same species, and/or different and/or distinct organisms. Inone non-limiting example, the HAMs are from one or more extremophiles,such as, but not limited to: hyperthermophilic, thermophilic,acidophilic, thermoacidophilic, and/or polyextremophilic organisms. Somenon-limiting examples of organisms include: Alicyclobacillusacidocaldarius; Clostridium thermocellum; Clostridium thermolacticum;Clostridium thermohydrosulfuricum; Trichoderma reesei; a variety ofBacillus species (i.e., B. subtilis, B. thermoamylovorans, B.stearothermophilus, B. granulobacter, B. pectinovorum, B. halodurans,etc.); Moorella thermoautotrophica; Moorella thermoacetica;Streptococcus (Lactobacillus) thermophilus; and/or other extremophilicbacteria that possess one or more nucleic acids associated with thebreakdown and/or hydrolysis of biomass and/or lignocellulosic materialinto more simple sugar molecules and/or sugar monomers. It is alsocontemplated that the HAMs may be isolated from, derived from, orotherwise originate from any type and/or kind of organism that includesone or more of the HAMs and/or other extremophilic bacteria that possessone or more nucleic acids encoding proteins involved in breakdown and/orhydrolysis of biomass and/or lignocellulosic material into products. Itis also contemplated that the HAMs may be isolated from, derived from,or otherwise originate from any type and/or kind of organism thatincludes one or more of the HAMs, such as, but not limited to, thosefound online at wzw.tum.de/mbiotec/cellmo.htm; or available from aninstitution, such as, but not limited to, the Advanced BiotechnologyCenter (ABC) (Italy); Interlab Cell Line Collection (BiotechnologyDept.) (Italy); the Australian Government Analytical Laboratories(AGAL); the American Type Culture Collection (ATCC); the BelgianCoordinated Collections of Microorganisms (BCCM); the Prime Minister'sServices Federal Office for Scientific, Technical and Cultural Affairs(OSTC) (Belgium); Bureau of Microbiology at Health Canada (BMHC);Centraalbureau voor Schimmelcultures (CBS) (the Netherlands); ChinaCenter for Type Culture Collection (CCTCC) (Wuhan); China Committee forCulture Collection of Microorganisms (Beijing); Coleccion Espafiola deCultivos Tipo (CECT) (Spain); Collection Nationale De Cultures DeMicroorganismes (CNCM) (Institut Pasteur, France); Collection ofIndustrial Yeasts DBVPG (Perugia, Italy); Culture Collection of Algaeand Protozoa (CCAP) (United Kingdom); Culture Collection of Yeasts (CCY)(Slovakia); Czech Collection of Microorganisms (CCM); Deutsche Sammlungvon Mikroorganismen und Zellkulturen GmbH (DSMZ) (Germany); EuropeanCollection of Cell Cultures (ECACC) (United Kingdom); Institute ofAgriculture and Food Biotechnology (IAFB) Collection of IndustrialMicroorganisms (Poland); International Mycological Institute (IMI)(United Kingdom); International Patent Organism Depositary (IPOD)(Tsukuba, Japan); Korean Cell Line Research Foundation (KCLRF); KoreanCollection for Type Cultures (KCTC); Korean Culture Center ofMicroorganisms (KCCM); Microbial Strain Collection of Latvia (MSCL);National Bank for Industrial Microorganisms and Cell Cultures (NBIMCC)(Bulgaria); National Collection of Agricultural and IndustrialMicroorganisms (NCAIM) (Budapest, Hungary); National Collection of TypeCultures (NCTC) (United Kingdom); National Collection of Yeast Cultures(NCYC); AFRC Institute of Food Research (United Kingdom); NationalCollections of Industrial, Food and Marine Bacteria (NCIMB) (Scotland);National Research Center of Antibiotics (Moscow); Polish Collection ofMicroorganisms (PCM) (Poland); Russian Collection of Microorganisms(VKM); and the Russian National Collection of Industrial Microorganisms(VKPM) (Russia).

In embodiments, one or more of the HAMs may be incorporated into,integrated into, and/or otherwise expressed in one or more organismsthat may already have the ability to ferment sugars to products. Suchorganisms may be any organism known in the art that has the ability toferment sugars to alcohols or other products. A non-exhaustive list oforganisms may include: Clostridium thermocellum; Clostridiumthermolacticum; Clostridium thermohydrosulfuricum; a variety of Bacillusspecies (i.e., B. subtilis, B. thermoamylovorans, B. stearothermophilus,B. granulobacter, B. pectinovorum, B. halodurans, etc.); Moorellathermoautotrophica; Moorella thermoacetica; Streptococcus(Lactobacillus) thermophilus; Saccharomyces cerevisiae (yeast),Zymomonas mobilis, Candida shehatae, E. coli, those found at any of theweb sites or institutions listed herein, and/or other organisms thatpossess one or more nucleic acids coding for proteins involved infermentation of sugars to products.

It is additionally contemplated that the organism able to ferment sugarsto products may be any organism that has nucleic acids encoding for oneor more proteins involved with the fermentation pathways, such as, butnot limited to, hexokinase I, hexokinase II, glucokinase,glucose-6-phosphate isomerase, phosphofructokinase, aldolase,triosephosphate isomerase, glyceraldehyde-3-phosphate dehydrogenase,3-phosphoglycerate kinase, phosphoglycerate mutase, enolase, enolase I,pyruvate decarboxylase, citrate synthase, aconitase, isocitratedehydrogenase, succinate dehydrogenase, fumarase, xylose reductase,xylitol dehydrogenase, xylulokinase, phosphoketolase, lactatedehydrogenase, acetyl-CoA-acetyl transferase, β-hydroxybutyryl-CoAdehydrogenase, crotonase, butyryl CoA dehydrogenase, phosphobutyrylase,butyrate kinase, and alcohol dehydrogenase. In embodiments of thepresent invention, the organism may already include those enzymes and/ornucleic acids encoding enzymes, necessary for fermenting sugars toproducts. Some non-limiting examples of these enzymes include:hexokinase I, hexokinase II, glucokinase, glucose-6-phosphate isomerase,phosphofructokinase, aldolase, triosephosphate isomerase,glyceraldehyde-3-phosphate dehydrogenase, 3-phosphoglycerate kinase,phosphoglycerate mutase, enolase, enolase I, pyruvate decarboxylase,citrate synthase, aconitase, isocitrate dehydrogenase, succinatedehydrogenase, fumarase, xylose reductase, xylitol dehydrogenase,xylulokinase, phosphoketolase, lactate dehydrogenase, acetyl-CoA-acetyltransferase, 3-hydroxybutyryl-CoA dehydrogenase, crotonase, butyryl CoAdehydrogenase, phosphobutyrylase, butyrate kinase, and alcoholdehydrogenase.

In embodiments, one or more of the HAMs may include and/or be expressedwith one or more regulatory elements and/or proteins, such as, but notlimited to, promoters, enhancers, transcription factors, activators,silencers, and/or so forth. Non-limiting examples of such regulatoryelements may be found in the patent applications and patents previouslyincorporated herein by reference. In this manner, the transcriptionand/or expression of one or more of the HAMs may be regulated and/orcontrolled. In a non-limiting example, the HAMs may include a promoterregion substantially similar to promoter regions of one or more of theorganism's Fermentation Associated Molecules (FAMs) (those nucleic acidsencoding proteins involved in the fermentation of sugars to products).In this manner, transcription and/or expression of one or more of theHAMs occur at substantially the same time, or substantially simultaneouswith, the transcription and/or expression of one or more of the FAMs. Itis also contemplated that one or more of the HAMs may be included and/orintegrated under the control of the same promoter and/or promoterregions of the FAMs in one or more of the organisms. Some non-limitingexamples of types of promoters contemplated include: constitutivepromoters, tissue specific or development stage promoters, induciblepromoters, and/or synthetic promoters. In one embodiment, the promotermay be a standard promoter used in expression vectors, such as, but notlimited to, the T7 and Sp6 Phage promoters, which promote the expressionof inserted nucleic acids in a phage type vector.

In embodiments, one or more of the HAMs may comprise and/or be includedin a delivery system that may deliver, incorporate, transfer, and/orassist in the delivery and/or expression of one or more of the HAMs intoone or more of the organisms able to break down and/or convert celluloseto more simple sugar molecules. The delivery system may comprise anymethod for incorporating DNA, nucleic acids and/or vectors in cellsknown in the art, such as but not limited to, transfection,electroporation, lipofection, transformation, gene guns, BiolisticParticle Delivery Systems, and/or so forth.

It is additionally contemplated that one or more HAMs may beincorporated into any type and/or kind of vector known in the art. Anon-exhaustive list of potential vectors include: plasmids,bacteriophages, viruses, yeast artificial chromosomes (YACs) bacterialartificial chromosomes (BACs), P1 bacteriophage-derived chromosomes(PACs), mammalian artificial chromosomes, and/or so forth. The insertionand/or incorporation of desired nucleic acids into vectors is wellunderstood, and in many instances vectors and/or insertion of nucleicacids into vectors are commercially available (i.e., through Empiregenomics at empiregenomics.com/site/products_bacclones.php).

In embodiments, the delivery systems and/or vectors that may be used toincorporate one or more of the HAMs may include selective markers, tags,or the like. Such selective markers tags, etc., may enable thedetermination of a successful delivery, integration, and/or expressionof a vector including one or more of the HAMs. Some non-limitingexamples of selective markers, tags, etc., include: antibioticresistance, amino acid/nutrient markers, color markers, fluorescentmarkers (i.e., GFP, RFP, etc.), His-tags, FLAG tags, Calmodulin-tags,HA-tags, Maltose binding protein-tags, Thioredoxin-tags, S-tags,Strep-tags, Nus-tags, and/or so forth.

In embodiments, it is contemplated that one or more of the HAMs may beintegrated with, incorporated into, and/or become part of the genomeand/or DNA of the host organism. Integrating one or more of the HAMsinto the host DNA may provide for better expression, replication tosubsequent host progeny, and/or protection from DNA degradation by thehost organism. Numerous methods and/or vectors for integrating,inserting, and/or incorporating a desired nucleic acid into a hostgenome are well known in the art. A non-exhaustive list of potentialvectors include: phage lambda (λ), adeno-associated virus (AAV),adenovirus, lentivirus, retroviruses, transposons, or the like.Additionally, a multipurpose vector system may be used as taught inLaitinen et al., A multi-purpose vector system . . . , Nucleic AcidsResearch, 2005, vol. 33, no. 4, which is incorporated by referenceherein.

Expression/Integration of Fermentation Associated Molecules

In one embodiment of the invention one or more Fermentation AssociatedMolecules (FAMs), encoding for proteins associated with, involved in,and/or assisting in the fermentation of sugars to products may beincorporated and/or inserted into an organism that has the ability tobreak down lignocellulosic materials to more simple sugar molecules. TheFAMs may include any nucleic acids, proteins and/or enzymes that areassociated with the fermentation of sugars to products. A non-exhaustivelist of these proteins and/or enzymes includes: hexokinase I, hexokinaseII, glucokinase, glucose-6-phosphate isomerase, phosphofructokinase,aldolase, triosephosphate isomerase, glyceraldehyde-3-phosphatedehydrogenase, 3-phosphoglycerate kinase, phosphoglycerate mutase,enolase, enolase I, pyruvate decarboxylase, citrate synthase, aconitase,isocitrate dehydrogenase, succinate dehydrogenase, fumarase, xylosereductase, xylitol dehydrogenase, xylulokinase, phosphoketolase, lactatedehydrogenase, acetyl-CoA-acetyl transferase, β-hydroxybutyryl-CoAdehydrogenase, crotonase, butyryl CoA dehydrogenase, phosphobutyrylase,butyrate kinase, and alcohol dehydrogenase.

In additional embodiments, the FAMs encompass all those nucleic acidscoding for proteins associated with the replication, transcription,translation, and/or expression of nucleic acids associated with thefermentation of sugars to a product. In a non-limiting example, FAMs maycomprise those nucleic acids constituting either regulatory sequences orcoding for proteins involved with the regulation of FAMs, such as, butnot limited to, those nucleic acid sequences and/or proteins thatincrease replication, transcription, expression, etc., of FAMs. Further,FAMs may comprise those nucleic acids that are involved with thedown-regulation of FAMs, either directly or through the proteins theymay encode. A non-exhaustive list of proteins encoded by these FAMs mayinclude: transcription factors, enhancers, repressors, DNA bindingproteins, or the like.

In further embodiments, the FAMs may include derivatives, analogs,and/or modified versions of one or more FAMs. These derivatives,analogs, and/or modified versions may include those modificationscarried out either in vive or in vitro. Some non-limiting examples ofderivatives, analogs, and/or modifications include: substitutions,deletions, mutations, modifications, etc. Modifications may comprise allmodifications occurring to nucleic acids, DNA, RNA, proteins, etc., suchas, but not limited to, acetylation, deacetylation, alkylation,methylation, demethylation, carboxylation, glycosylation,phosphorylation, hydroxylation, PEGylation, biotinylation, and/or anyother type of modification known in the art.

In yet other embodiments, the FAMs may encompass those nucleic acids,regulatory sequences, proteins, and/or enzymes which are encoded by theFAMs that are derivatives of FAMs and/or include tags, markers, and/orother modifications. A non-exhaustive list of tags may include:His-tags, FLAG tags, Calmodulin-tags, HA-tags, Maltose bindingprotein-tags, Thioredoxin-tags, S-tags, Strep-tags, Nus-tags, or thelike. The FAMs may also include those nucleic acids, proteins, and/orenzymes that include fluorescent markers, such as, but not limited to,GFP, RFP, YFP, BFP, or the like.

Advantageously, the incorporation of the FAMs into the one or moreorganisms with the ability to break down biomass to more simple sugarmolecules may enable the one or more genetically modified organisms toconvert lignocellulose and/or other biomass materials more efficientlyinto a product under a variety of pH and/or temperature conditions. Inone embodiment, the one or more genetically modified organisms may carryout the biomass conversion in a range of pH conditions from at or lessthan a pH of about 7; at or less than a pH of about 5, from about a pHof 1 to about a pH of 5, and/or from about a pH of 1 to about a pH of1.3.

Advantageously, in one embodiment, the ability of the geneticallymodified organism to function and/or convert biomass into products at pHconditions of 1 to 5, eliminates the need to neutralize and/or increasethe pH to a range of about 5 to 6.0 for enzymatic hydrolysis andfermentation, which, using conventional methods, is necessary for thehydrolysis and fermentation of cellulose to products. Indeed, inconventional methods of dilute acid hydrolysis, the pretreatmenthydrolysate needs to be neutralized to enable enzymatic hydrolysis andfermentation to occur. As such, in one embodiment, the FAMs may code forenzymes which comprise thermophilic, thermoacidophilic, and/oracidophilic properties and/or characteristics, or in the alternative maybe derived from, analogs of, and/or homologues of one or morethermophilic, thermoacidophilic, and/or acidophilic FAMs from otherorganisms.

It is additionally contemplated that one or more of the proteins encodedby the FAMs may function and/or have an optimum pH range of above a pHof 5. Indeed, one or more of the proteins encoded by the FAMs mayfunction and/or enable fermentation of sugar to products in pHconditions that range from a pH of about 5 to a pH of about 14.

In still another embodiment, enzymes encoded by one or more FAMs mayfunction to and/or assist in fermenting sugars to products in a broadrange of temperatures. Conventional methods and techniques ofpretreatment using dilute acid hydrolysis and low temperature acidhydrolysis occur at temperature ranges of about 160 degrees Celsius anda range of eighty (80) degrees to one hundred (100) degrees Celsius,respectively. However, using the conventional methods, in order to beginthe enzymatic hydrolysis and fermentation reactions, the pretreatmentmixture/slurry needs to be cooled to around forty (40) degrees to fifty(50) degrees Celsius.

In contrast to the current and/or conventional methods, the FAMsincorporated into one or more of the organisms described herein mayfunction to, assist in, and/or carry out the fermentation process in abroad range of temperatures. Some non-limiting examples of temperatureranges include: at least about fifty (50) degrees Celsius, at leastabout seventy (70) degrees Celsius, from about forty-five (45) degreesCelsius to about eighty (80) degrees Celsius; from about eighty (80)degrees Celsius to about one hundred (100) degrees Celsius; from abouteighty-five (85) degrees to about ninety-five (95) degrees Celsius;and/or from about ninety (90) degrees to about one hundred (100) degreesCelsius.

In further embodiments, the one or more FAMs may be isolated from,derived from and/or originate from one or more organisms. The one ormore organisms may comprise the same organism, organisms within the samegenus and/or within the same species, and/or different and/or distinctorganisms. In one non-limiting example, the FAMs are from one or moreextremophiles, such as, but not limited to: hyperthermophilic,thermophilic, acidophilic, thermoacidophilic, and/or polyextremophilicorganisms. Some non-limiting examples of organisms include:Alicyclobacillus acidocaldarius, Clostridium thermocellum; Clostridiumthermolacticum; Clostridium thermohydrosulfuricum; a variety of Bacillusspecies (i.e., B. subtilis, B. thermoamylovorans, B. stearothermophilus,B. granulobacter B. pectinovorum, B. halodurans, etc.); Moorellathermoautotrophica; Moorella thermoacetica; Streptococcus(Lactobacillus) thermophilus, those found at any of the web sites orinstitutions listed herein, and/or other organisms that possess one ormore nucleic acid sequences involved in fermentation of sugars toproducts. It is also contemplated that the FAMs may be isolated from,derived from, or otherwise originate from non-extremophilic organisms,such as, but not limited to, Saccharomyces cerevisiae (yeast), Zymomonasmobilis, Candida shehatae, E. coli, and/or other organisms which possessone or more nucleic acids involved in fermentation of sugars toproducts.

In embodiments, one or more of the FAMs may be incorporated into,integrated into, and/or otherwise expressed in one or more organismsthat already have the ability to break down lignocellulose and/orbiomass to more simple sugar molecules. Such organisms may be anyorganism known in the art that has the ability to break downlignocellulose to more simple sugar molecules. A non-exhaustive list ofsuch organisms may include: Alicyclobacillus acidocaldarius; Clostridiumthermocellum; Clostridium thermohydrosulfuricum; and/or any otherorganism that is known to have the ability to break down lignocelluloseand/or biomass. It is additionally contemplated that the organism ableto break down lignocellulose and/or biomass may be any organism that hasnucleic acid sequences encoding one or more types of endo- and/orexo-cellulases, i.e., endo-beta-1,4-glucanase; one or more types ofhemicellulases, i.e., endo-beta-1,4-xylanase; and/or one or morexylosidase enzymes.

In one embodiment of the present invention, an organism may alreadyinclude enzymes and/or nucleic acids encoding enzymes, for breaking downat least one of the three major components of lignocellulose: cellulose,hemicellulose, and lignin. Some non-limiting examples of these enzymesinclude endo- and/or exo-β-1,4-glucanases. The endo- and/orexo-β-1,4-glucanases may function to hydrolyze the linked glucoseresidues (endo activity) and/or hydrolyze the ends of the cellulosepolymers (exo activity). Additionally, the organism may already includeendo-β-1,4-xylanase; and/or one or more xylosidase enzymes, such enzymespossessing the ability to hydrolyze one or more types of hemicellulosepolymers. In an additional embodiment, the organism may includeadditional and/or accessory enzymes, proteins, or nucleic acid sequencesthat may assist in completely hydrolyzing, transporting, metabolizingand/or breaking down the one or more types of lignocellulose polymersinto more simple sugar molecules and/or sugar monomers.

In embodiments, it is contemplated that the organism may include one ormore enzymes and/or nucleic acids encoding enzymes that have the abilityto break down, transport, metabolize, and/or hydrolyze lignin molecules;including nucleic acids encoding for ligninolytic enzymes such asmanganese peroxidase, laccase, lignin peroxidase, or the like.

In embodiments, one or more of the FAMs may include and/or be expressedwith one or more regulatory factors, regulatory elements, and/orproteins, such as, but not limited to, promoters, enhancers,transcription factors, activators, silencers, and/or so forth.Non-limiting examples of such regulatory elements may be found in thepatent applications and patents previously incorporated herein byreference. In this manner, the transcription and/or expression of one ormore of the FAMs may be regulated and/or controlled. In a non-limitingexample, the FAMs may include a promoter region substantially similar topromoter regions of one or more of the organism's Hydrolysis AssociatedMolecules (HAMs) (those nucleic acids involved in breaking downlignocellulosic materials to simpler sugars). In this manner,transcription and/or expression of one or more of the FAMs occur atsubstantially the same time, or substantially simultaneous with, thetranscription and/or expression of the Hydrolysis Associated Molecules.It is also contemplated one or more of the FAMs may be included and/orintegrated under the control of the same promoter and/or promoterregions of the Hydrolysis Associated Molecules in one or more of theorganisms. Some non-limiting examples of types of promoters contemplatedinclude: constitutive promoters, tissue specific or development stagepromoters, inducible promoters, and/or synthetic promoters. In oneembodiment, the promoter may be a standard promoter used in expressionvectors, such as, but not limited to, the T7 and Sp6 Phage promoters,which promote the expression of inserted nucleic acids in a phage typevector.

In embodiments, one or more of the FAMs may comprise and/or be includedin a delivery system that may deliver, incorporate, transfer, and/orassist in the delivery and/or expression of one or more of the FAMs intoone or more of the organisms able to break down and/or convertlignocellulose to more simple sugar molecules. The delivery system maycomprise any method for incorporating DNA, nucleic acids and/or vectorsinto cells known in the art. A non-exhaustive list of methods mayinclude: transfection, electroporation, lipofection, transformation,gene guns, Biolistic Particle Delivery System, and/or so forth.

It is additionally contemplated that one or more FAMs may beincorporated into any type and/or kind of vector known in the art. Anon-exhaustive list of potential vectors include: plasmids,bacteriophages, viruses, yeast artificial chromosomes (YACs), bacterialartificial chromosomes (BACs), P1 bacteriophage-derived chromosomes(PACs), mammalian artificial chromosomes, and/or so forth. The insertionand/or incorporation of desired nucleic acids into vectors is wellunderstood in the art, and in many instances vectors and/or insertion ofnucleic acids into vectors are commercially available (i.e., throughEmpire genomics empiregenomics.com/site/products_bacclones.php).

In embodiments, the delivery systems and/or vectors that may be used toincorporate one or more of the FAMs may include and/or encode selectivemarkers, tags, or the like, that may enable the determination of asuccessful delivery, integration, and/or expression of a vectorincluding one or more of the FAMs. Some non-limiting examples ofselective markers, tags, etc., include: antibiotic resistance, aminoacid/nutrient markers, color markers, fluorescent markers (i.e., GFP,RFP, etc.), His-tags, FLAG tags, Calmodulin-tags, HA-tags, Maltosebinding protein-tags, Thioredoxin-tags, S-tags, Strep-tags, Nus-tags,and so forth.

In embodiments, it is contemplated that one or more of the FAMs may beintegrated with, incorporated into, and/or become part of the genomeand/or DNA of the host organism. Integrating one or more of the FAMsinto the host DNA may provide for better expression, replication tosubsequent host progeny, and/or protection from DNA degradation by thehost organism. There are numerous methods and/or vectors forintegrating, inserting, and/or incorporating a desired nucleic acid intoa host genome which are well known in the art. A non-exhaustive list ofpotential vectors include: phage lambda (λ), adeno-associated virus(AAV), adenovirus, lentivirus, retroviruses, transposons, or the like.Additionally, a multipurpose vector system may be used as taught inLaitinen et al., A multi-purpose vector system . . . , Nucleic AcidsResearch, 2005, vol. 33, no. 4, which is incorporated by referenceherein.

BioProcessing

In embodiments, one or more of the genetically modified organismsdescribed herein may produce one or more proteins from the FAMs and/orHAMs intracellularly and/or extracellularly. In producing proteinsextracellularly, the genetically modified organism may excrete theproteins, enzymes, etc., into the extracellular and/or surroundingenvironment. It is additionally contemplated that the outer membraneand/or cell wall of the genetically modified organism may be coated withand/or include one or more proteins encoded by the HAMs. In this manner,breakdown and/or hydrolysis of biomass/lignocellulosic material isrealized in the extracellular environment.

In embodiments, one or more of the genetically modified organismsdescribed herein may convert biomass and/or lignocellulosic material toproducts at least partially intracellularly, or otherwise internal tothe genetically modified organism. In a non-limiting example, at leastpartially hydrolyzed sugar molecules may be brought and/or transportedinto the genetically modified organism and fermented to ethanol oranother product. The one or more genetically modified organism may thenexcrete the ethanol or other product into the extracellular environment.

It is contemplated that one more of the genetically modified organismsdescribed herein may have multiple methods of use, uses, and/orapplications in a wide variety of industries, laboratories, markets,etc.

In embodiments, one or more of the genetically modified organismsdescribed herein may be applied to biomass and/or lignocellulosicmaterial such that the biomass and/or lignocellulosic material is brokendown, depolymerized, and subsequently converted to products. The one ormore genetically modified organisms may be applied to the biomass orlignocellulosic material/composition in any manner known in the art. Ina non-limiting example, the biomass and/or lignocellulosic material maybe ground up and/or undergo a grinding process. Additionally, thebiomass and/or lignocellulosic material may undergo variouspretreatments prior to exposure to the recombinant organism. Suchtreatments may include, but are not limited to, sulfur dioxide, steamexplosion, acid hydrolysis, ammonia hydrolysis, autohydrolysis, or thelike. Additional, non-limiting examples are described in Thomas et al.,which is incorporated by reference herein.

In embodiments, one or more of the genetically modified organismsdescribed herein may be applied to biomass and/or lignocellulosicmaterial, such as, but not limited to, cellulose, through the use ofmultiple methods and/or procedures. In one non-limiting example, alignocellulosic composition may comprise one or more solidsubstrates/phases which are inoculated with the one or more geneticallymodified organisms, and/or cultures thereof. Additionally, thelignocellulosic composition may be embodied in a liquid, orsubstantially liquid substrate/phase; and the one or more geneticallymodified organisms introduced therein.

In embodiments, it is additionally contemplated that enzymes and/orproteins encoded by any and/or all of the HAMs and/or FAMs may beextracted, removed and/or isolated from one or more genetically modifiedorganisms described herein. These enzymatic and/or proteinaceouscompositions may then be applied to biomass and/or lignocellulosicmaterial.

In embodiments, the biomass and/or lignocellulosic composition isinoculated with a genetically modified organism culture. It iscontemplated there may be a variety of conditions (i.e., pH,concentrations of biomass and/or lignocellulosic material, concentrationof genetically modified organisms, temperature, pressure, etc.) at whichhydrolysis of the biomass and/or lignocellulosic material and subsequentfermentation may occur. Indeed, the pH of the hydrolysis may includeranges of at or less than a pH of about 7; at or less than a pH of about5; about a pH of 1 to about a pH of 5; and/or from about a pH of 1 toabout a pH of 1.3.

In embodiments, the ability of the one or more hydrolysis and/orfermentation reactions occurring in pH conditions of 1 to 5, mayeliminate the need to neutralize and/or increase the pH to a range ofabout 5 to 6 following acid pretreatment for enzymatic hydrolysis andfermentation that, using conventional methods, is necessary for thehydrolysis and fermentation of lignocellulosic material to products.Indeed, in conventional methods of dilute acid hydrolysis, thepretreatment hydrolysate needs to be neutralized to enable enzymatichydrolysis and fermentation to occur. As such, in one embodiment, theHAMs may code for enzymes that comprise thermophilic, thermoacidophilic,and/or acidophilic properties and/or characteristics, or in thealternative may be derived from one or more thermophilic,thermoacidophilic, and/or acidophilic organisms.

It is additionally contemplated that one or more hydrolysis and/orfermentation reactions may occur and/or have an optimum pH range ofabove a pH of 5. Indeed, the hydrolysis and/or fermentation reactionsmay occur in pH conditions that range from a pH of about 5 to a pH ofabout 14.

In embodiments, the one or more hydrolysis and/or fermentation reactionsmay occur in a broad range of temperatures. Some non-limiting examplesof temperature ranges include: from about fifty (50) degrees Celsius;from about seventy (70) degrees Celsius; from about eighty (80) degreesCelsius to about one hundred (100) degrees Celsius; from abouteighty-five (85) degrees to about ninety-five (95) degrees Celsius;and/or from about ninety (90) degrees to about one hundred (100) degreesCelsius.

In embodiments, one or more of the genetically modified organismsdescribed herein may be grown and/or cultured in accordance with knownmethods. Such genetically modified organism cultures may be fluidlyapplied and/or contacted with biomass and/or lignocellulosiccompositions, such as, but not limited to, polysaccharides, cellulose,lignocellulose, hemicellulose, lignin, starch, sugars, sugar oligomers,carbohydrates, complex carbohydrates, chitin, heteroxylans, glycosides,xylan-, glucan-, galactan-, or mannan-decorating groups.

In embodiments, cellular and/or proteinaceous extracts of thegenetically modified organism cultures may be isolated. Protein and/orcellular isolations and extractions may be conducted throughconventional methods. The cellular and/or proteinaceous extracts may beapplied to and/or contacted with the biomass and/or lignocellulosiccompositions as described herein.

In embodiments, one or more of the genetically modified organisms and/orone or more of the cellular and/or proteinaceous extracts describedherein may be applied and/or fluidly contacted with biomass and/orlignocellulosic compositions under thermophilic, acidophilic, and/orthermoacidophilic conditions. In a non-limiting example, the temperaturemay be at or above about fifty (50) degrees and/or the pH at or belowabout the pH of 5 in a treatment mixture, slurry, apparatus or otherarea in which the fluid application occurs.

It is contemplated that a variety of conventional methods and/ortechniques may be used to create the one or more genetically modifiedorganisms described herein. One non-limiting method is described in U.S.Pat. No. 4,624,922, which is incorporated by reference herein.

Integration of Pretreatment, Biological Processing, and Product Recovery

Typically, the fermentative biological production of products frombiomass proceeds through four distinct steps: pretreatment, cellulolytichydrolysis, fermentation, and recovery. The pretreatment and recoverysteps remain distinct in typical process designs, while the cellulolytichydrolysis and fermentations steps can be separate steps or can becombined in various ways.

The pretreatment step is typically a physical or chemical pretreatment,such as an acid pretreatment, that effects a modification of the biomassto aid in further processing with cellulase enzymes. Typically, after asuitable period, the acid of the pretreatment is neutralized beforeinitiating cellulolytic hydrolysis. In the most basic processingconfiguration, cellulolytic hydrolysis is carried out using exogenouslyadded enzymes and is carried out separately from the fermentation step(separate hydrolysis and fermentation, or SHF). Glucose generated fromthe cellulose during cellulolytic hydrolysis, and pentose sugarsreleased during the pretreatment, are separately consumed by fermentingorganism(s) that are introduced to ferment the simple sugars intoethanol or another product of value. After fermentation, the product isrecovered in a recovery step.

Additional processing configurations are typically utilized in which thecellulolytic hydrolysis using exogenously added enzymes and fermentationsteps may be integrated. These include separate cellulolytic hydrolysisfollowed by co-fermentation of the pentose and hexose sugars (separatehydrolysis and co-fermentation, or SHCF), simultaneous cellulolytichydrolysis and glucose fermentation (simultaneous saccharification andfermentation, or SSF) with separate pentose fermentation, simultaneouscellulolytic hydrolysis and co-fermentation of the glucose and pentosesugars (simultaneous saccharification and co-fermentation, or SSCF).

A final processing configuration can be used in which the organismutilized to ferment glucose released during cellulolytic hydrolysisserves as the source of the cellulolytic enzymes. In this configuration,a physical and/or chemical pretreatment is utilized to improve theaction of the endogenously produced cellulolytic enzymes on thecellulose. This processing configuration is referred to as ConsolidatedBioprocessing (CBP).

In embodiments of the invention, the above schemes may be modifiedthrough the use of enzymes and/or organisms active and stable atincreased temperatures and decreased pH to allow the combination ofpretreatment and/or product recovery with the conventional biologicalprocessing steps. In certain embodiments, the pretreatment may beconducted at lowered temperatures relative to existing dilute acidpretreatment technology and this step may be carried out enzymaticallyutilizing thermoacidophilic lignocellulose-degrading enzymes. Thealtered pretreatment and subsequent biological processing (cellulolytichydrolysis and fermentation) may be carried out sequentially or combinedtogether. The pretreatment and biological processing, may, in certainembodiments, be carried out by isolated enzymes and/or organisms for thecellulolytic hydrolysis and/or fermentation steps according to thepresent invention. The enzymes may be added exogenously or may beproduced by the fermentation organisms in a Combined Pretreatment andConsolidated Bioprocessing (CPBP) configuration. In certain embodiments,the thermoacidophilic enzymes and organisms may include Alicyclobacillusacidocaldarius, genetically modified organisms comprising one or morenucleotides sequences derived or isolated from Alicyclobacillusacidocaldarius, or by extracts and/or lysates comprising one or moreproteins produced by or derived from Alicyclobacillus acidocaldarius. Inembodiments, the pretreatment step may comprise an acid pretreatment;alkaline pretreatment; hydrothermal pretreatment; and/or anorganosolvent pretreatment.

In further embodiments, the acid or alkali added to or generated duringa pretreatment step may not need to be neutralized before hydrolysisand/or fermentation. In embodiments, the non-neutralization of thepretreatment conditions may lead to the decreased function or death ofany unwanted organisms present on the incoming biomass to be treated. Inembodiments, the non-neutralization of the pretreatment conditions mayresult in decreased byproducts or unwanted products as the function ofany unwanted organisms present on the incoming biomass to be treated maybe decreased.

In additional embodiments, one or more genetically modified organisms,HAMs, and/or FAMs according to the present invention may play a role inthe pretreatment, cellulolytic hydrolysis, and fermentation, or in anycombination thereof. For example, a genetically modified organismaccording to the present invention may carry out processes contributingto pretreatment and cellulolytic hydrolysis, and a second geneticallymodified organism according to the present invention may separately orsimultaneously carry out the fermentation. In another non-limitingexample, thermoacidophilic enzymes according to the present inventionmay carry out processes contributing to pretreatment, and one or moregenetically modified organism(s) according to the present invention maycarry out processes contributing to cellulolytic hydrolysis and/orfermentation. In a further non-limiting example, a single geneticallymodified organism(s) according to the present invention may carry outprocesses contributing to pretreatment, cellulolytic hydrolysis, andfermentation.

In embodiments, a reduced severity acid pretreatment step may becombined with, or occur sequentially with, the addition ofthermoacidophilic lignocellulose-degrading enzymes, and occur separatelyor concurrently with one or both of the cellulolytic hydrolysis andfermentation steps.

Expression/Integration of Hydrolysis Associated Molecules andFermentation Associated Molecules

In embodiments of the invention one or more Hydrolysis AssociatedMolecules (HAMs) and/or one or more Fermentation Associated Molecules(FAMs) may be incorporated and/or inserted into an organism.

The incorporation of the HAMs and FAMs into the one or more organismsmay enable the one or more organisms to convert lignocellulosic and/orother biomass materials into a product at a variety of pH andtemperature conditions. In one embodiment, the one or more geneticallymodified organisms may carry out the biomass conversion in a range of pHconditions from at or less than a pH of about 7; at or less than a pH ofabout 5; about a pH of 1 to about a pH of 5; and/or from about a pH of 1to about a pH of 1.3.

In embodiments, the ability of the organism(s) to function and/orconvert biomass into products in pH conditions of 1 to 5, may eliminatethe need to neutralize and/or increase the pH to a range of about 5 to 6for enzymatic hydrolysis and/or fermentation after dilute acidpretreatment, which, using conventional methods, is necessary for thehydrolysis and fermentation of cellulose to products. Indeed, inconventional methods of dilute acid hydrolysis, the pretreatmenthydrolysate needs to be neutralized to enable enzymatic hydrolysis andfermentation to occur. As such, in one embodiment, the HAMs and FAMs maycode for or be enzymes that possess thermophilic, thermoacidophilic,and/or acidophilic properties and/or characteristics, or in thealternative may be derived from one or more thermophilic,thermoacidophilic, and/or acidophilic organisms.

It is additionally contemplated that one or more of the HAMs and FAMsmay function and/or have an optimum pH range of above a pH of 5. Indeed,one or more of the HAMs and FAMs may function and/or enable breakdownand/or hydrolysis of biomass and/or lignocellulosic material intoproducts in pH conditions that range from a pH of about 5 to a pH ofabout 14.

Conventional methods and techniques of pretreatment for dilute acidhydrolysis and low temperature acid hydrolysis occur at temperatureranges of about 160 degrees Celsius and a range of eighty (80) degreesto one hundred (100) degrees Celsius, respectively. However, using theconventional methods, in order to begin the enzymatic hydrolysis andfermentation reactions, the pretreatment mixture/slurry needs to becooled to around forty (40) degrees to fifty (50) degrees Celsius.

In contrast to the current and/or conventional methods, the HAMs andFAMs may function to, assist in, and/or carry out the breakdown and/orhydrolysis of biomass and/or lignocellulose into products in a broadrange of temperatures. Some non-limiting examples of temperature rangesinclude: at least about fifty (50) degrees Celsius; at least aboutseventy (70) degrees Celsius; from about forty-five (45) degrees Celsiusto about eighty (80) degrees Celsius; from about eighty (80) degreesCelsius to about one hundred (100) degrees Celsius; from abouteighty-five (85) degrees to about ninety-five (95) degrees Celsius;and/or from about ninety (90) degrees to about one hundred (100) degreesCelsius).

In embodiments, the one or more HAMs and FAMs may be isolated from,derived from and/or originate from one or more organisms. The one ormore organisms may comprise the same organism, organisms within the samegenus and/or within the same species, and/or different and/or distinctorganisms. In one non-limiting example, the HAMs and FAMs are from oneor more extremophiles, such as, but not limited to: hyperthermophilic,thermophilic, acidophilic, thermoacidophilic, and/or polyextremophilicorganisms. Some non-limiting examples of organisms include:Alicyclobacillus acidocaldarius; Clostridium thermocellum; Clostridiumthermolacticum; Clostridium thermohydrosulfuricum; Trichoderma reesei; avariety of Bacillus species (i.e., B. subtilis, B. thermoamylovorans, B.stearothermophilus, B. granulobacter B. pectinovorum, B. halodurans,etc.); Moorella thermoautotrophica; Moorella thermoacetica;Streptococcus (Lactobacillus) thermophilus; and/or other extremophilicorganisms that possess one or more nucleic acids encoding proteinsinvolved in breakdown and/or hydrolysis of biomass and/orlignocellulosic material and fermentation of sugars into products. It isalso contemplated that the HAMs and FAMs may be isolated from, derivedfrom, or otherwise originate from any type and/or kind of organism thatincludes one or more of the HAMs and FAMs, such as, but not limited to,those found at any of the web sites or institutions listed herein.

In embodiments, one or more of the HAMs and FAMs may be incorporatedinto, integrated into, and/or otherwise expressed in one or moreorganisms that do not have the ability to break down and/or hydrolyzebiomass and/or lignocellulose and/or ferment sugars to products. Suchorganisms may be any organism known in the art, such as, but not limitedto, E. coli, Thermus thermophilus, and/or any other suitable organismknown in the art.

In embodiments, one or more of the HAMs and FAMs may include and/or beexpressed with one or more regulatory elements and/or proteins, such as,but not limited to, promoters, enhancers, transcription factors,activators, silencers, and/or so forth. Non-limiting examples of suchregulatory factors may be found in the patent applications and patentspreviously incorporated herein by reference. In this manner, thetranscription and/or expression of one or more of the HAMs and FAMs maybe regulated and/or controlled. In a non-limiting example, the HAMs andFAMs may include a promoter region substantially similar to promoterregions of one or more of the organism's Fermentation AssociatedMolecules (those nucleic acids involved in the fermentation of sugars toproducts). In this manner, transcription and/or expression of one ormore of the HAMs and FAMs occur at substantially the same time, orsubstantially simultaneous with, the transcription and/or expression ofthe endogenous FAMs.

It is also contemplated that one or more of the HAMs and FAMs may beincluded and/or integrated under the control of the same promoter and/orpromoter regions in one or more of the organisms. Some non-limitingexamples of types of promoters contemplated include: constitutivepromoters, tissues specific or development stage promoters, induciblepromoters, and/or synthetic promoters. In one embodiment, the promotermay be a standard promoter used in expression vectors, such as, but notlimited to, the T7 and Sp6 Phage promoters, which promote the expressionof inserted nucleic acids in a phage type vector.

In embodiments, one or more of the HAMs and FAMs may comprise and/or beincluded in a delivery system that may deliver, incorporate, transfer,and/or assist in the delivery and/or expression of one or more of theHAMs and FAMs into one or more organisms. The delivery system maycomprise any method for incorporating DNA, nucleic acids and/or vectorsin cells known in the art, such as, but not limited to, transfection,electroporation, lipofection, transformation, gene guns, BiolisticParticle Delivery Systems, and/or so forth.

It is additionally contemplated that one or more HAMs and FAMs may beincorporated into any type and/or kind of vector known in the art. Anon-exhaustive list of potential vectors include: transposons, plasmids,bacteriophages, viruses, yeast artificial chromosomes (YACs), bacterialartificial chromosomes (BACs), P1 bacteriophage-derived chromosomes(PACs), mammalian artificial chromosomes, and/or so forth. The insertionand/or incorporation of desired nucleic acids into vectors is wellunderstood in the art, and in many instances vectors and/or insertion ofnucleic acids into vectors are commercially available (i.e., throughEmpire genomics at empiregenomics.com/site/products_bacclones.php).

In embodiments, the delivery systems and/or vectors that may be used toincorporate one or more of the HAMs and FAMs may include selectivemarkers, tags, or the like. Such selective marker and/or tags may enablethe determination of successful delivery, integration, and/or expressionof a vector including one or more of the HAMs and FAMs. Somenon-limiting examples of selective markers, tags, etc., include:antibiotic resistance, amino acid/nutrient markers, color markers,fluorescent markers (i.e., GFP, RFP, etc.), His-tags, FLAG tags,Calmodulin-tags, HA-tags, Maltose binding protein-tags,Thioredoxin-tags, S-tags, Strep-tags, Nus-tags, and/or so forth.

In embodiments, it is contemplated that one or more of the HAMs and FAMsmay be integrated with, incorporated into, and/or become part of thegenome and/or DNA of the host organism. Integrating one or more of theHAMs and FAMs into the host DNA may provide for better expression,replication to subsequent host progeny, and/or protection from DNAdegradation by the host organism. Numerous methods and/or vectors forintegrating, inserting, and/or incorporating a desired nucleic acid intoa host genome are well known in the art. A non-exhaustive list ofpotential vectors include: phage lambda (λ), adeno-associated virus(AAV), adenovirus, lentivirus, retroviruses, transposons, or the like.Additionally, a multipurpose vector system may be used as taught inLaitinen et al., 2005, A multi-purpose vector system . . . , NucleicAcids Research, vol. 33, no. 4, which is incorporated by referenceherein.

EXAMPLES Example 1: Delivery/Expression of Fermentation AssociatedMolecules in Alicyclobacillus acidocaldarius

Nucleic acids encoding enzymes involved in the fermentation of sugars(i.e., glucose, xylose, galactose, arabinose, mannose, etc.) to productsare provided to and subsequently expressed in Alicyclobacillusacidocaldarius using conventional molecular cloning techniques. Nucleicacids to be provided include those nucleic acids encoding for theproteins involved in the pathways that convert sugars to a product(FAMs). A non-exhaustive list of these proteins include: hexokinase I,hexokinase II, glucokinase, glucose-6-phosphate isomerase,phosphofructokinase, aldolase, triosephosphate isomerase,glyceraldehyde-3-phosphate dehydrogenase, 3-phosphoglycerate kinase,phosphoglycerate mutase, enolase, enolase I, pyruvate decarboxylase,citrate synthase, aconitase, isocitrate dehydrogenase, succinatedehydrogenase, fumarase, xylose reductase, xylitol dehydrogenase,xylulokinase, phosphoketolase, lactate dehydrogenase, acetyl-CoA-acetyltransferase, β-hydroxybutyryl-CoA dehydrogenase, crotonase, butyryl CoAdehydrogenase, phosphobutyrylase, butyrate kinase, and alcoholdehydrogenase. The FAMs may comprise nucleic acids from a wide varietyof organisms that use fermentation pathways. Some non-limiting examplesof these organisms include: Saccharomyces cerevisiae (yeast);Clostridium thermocellum; Clostridium thermohydrosulfuricum; a varietyof Bacillus species (i.e., B. subtilis, B. thermoamylovorans, B.stearothermophilus, B. granulobacter, B. pectinovorum, B. halodurans,and/or so forth); Alicyclobacillus acidocaldarius; Clostridiumthermolacticum; Trichoderma reesei; Moorella thermoautotrophica;Moorella thermoacetica; Streptococcus (Lactobacillus) thermophilus;and/or those found at any of the web sites or institutions listedherein.

Example 1(a): Delivery of FAMs Via a Transpososome

A transpososome is constructed to include nucleic acids encodingproteins involved in fermentation of sugars, or FAMs. Included in thetranspososome are the nucleic acids necessary for insertion of thetransposon, copies of the MuA transposase, Mu transposon ends, andprovision for expression of the FAMs included in the transpososome inthe particular organism in which they are to be utilized. Transpososomesmay also be constructed using sequences and elements optimized for usein gram-positive bacteria. See, Pajunen et al., Microbiology (2005) 151,1209-1218. Conventional techniques may be used to construct thetranspososome. Additionally, the construction and/or insertion ofdesired nucleic acids into transpososomes is well known in the art, see,Pajunen et al. It is contemplated that multiple transpososomes may beconstructed with FAMs and may include a variety of promoters, enhancersthat may function to enhance and/or regulate expression of FAMs.

Once constructed, the transpososomes are introduced intoAlicyclobacillus acidocaldarius. Transpososome constructs may beintroduced into A. acidocaldarius by electroporation, and so forth. Suchtechniques are known in the art and are detailed in Sambrook J. C., E.F. Fritsch, and T. Maniatas, 1989. Molecular Cloning: A LaboratoryManual, 2nd ed., Cold Spring Harbor Laboratory Press, N.Y., pp.6.3-6.34; or Sambrook and Russell, Molecular Cloning: A LaboratoryManual (3-Volume Set), 3^(rd) ed. (available online atmolecularcloning.com/); both references incorporated by referenceherein.

Example 2: Delivery/Expression of Hydrolysis Associated Molecules intoan Organism Able to Ferment Sugar to Ethanol

Nucleic acids encoding enzymes involved in the hydrolysis, or breakdown,of lignocellulosic materials (i.e., biomass, cellulose, hemicellulose,etc.) to simpler sugars (Hydrolysis Associated Molecules (HAMs)) aregenetically introduced by conventional molecular cloning techniques intoorganisms that ferment sugars to ethanol and subsequently expressedtherein. HAMs to be provided include those nucleic acids coding for theproteins involved in the pathways that break down or hydrolyzecellulose, hemicellulose, etc., to more simple sugar molecules. Anon-exhaustive list of these proteins include: esterases of thealpha-beta hydrolase superfamily; alpha-beta hydrolase;alpha-glucosidases; alpha-xylosidase; alpha-L-arabinofuranosidase;altronate hydrolase; a cellulose/endoglucanase; acellulase/endoglucanase; a polygalacturonase; an alpha-galactosidase; acellobiose phosphorylase; a glycogen debranching enzyme; an acetylesterase/acetyl hydrolase; a beta-1,4-xylanase; a cinnamoyl esterhydrolase; a carboxylesterase type B; abeta-galactosidase/beta-glucuronidase; a xylan alpha-1,2-glucuronidase;a 3-hydroxyisobutyryl-CoA hydrolase; a beta-glucosidase B-relatedglycosidase; and/or a chitooligosaccharide deacetylase, or as describedherein. The HAMs may comprise nucleic acids from a wide variety oforganisms that are able to hydrolyze and/or break down lignocellulosicmaterial to more simple sugar molecules.

Example 2(a): Delivery of HAMs Via a Transpososome

A transpososome is constructed to include nucleic acids encodingproteins involved in hydrolysis or breakdown of lignocellulosicpolymers, or HAMs. Included in the transpososome are the nucleic acidsnecessary for insertion of the transposon, copies of the MuAtransposase, Mu transposon ends, and provision for expression of theHAMs included in the transpososome in the particular organism in whichthey are to be utilized. Transpososomes may also be constructed usingsequences and elements optimized for use in gram-positive bacteria. See,Pajunen et al., Microbiology (2005) 151, 1209-1218. Conventionaltechniques may be used to construct the transpososome. Additionally, theconstruction and/or insertion of desired nucleic acids intotranspososomes is well known in the art, see, Pajunen et al. It iscontemplated that multiple transpososomes may be constructed with HAMsand may include a variety of promoters, enhancers that may function toenhance and/or regulate expression of HAMs.

Once constructed, the transpososomes are introduced into an organismthat is able to ferment and/or convert sugar molecules to a product. Anon-exhaustive list of potential organisms include: Clostridiumthermocellum; Clostridium thermohydrosulfuricum; a variety of Bacillusspecies (i.e., B. subtilis, B. thermoamylovorans, B. stearothermophilus,B. granulobacter, B. pectinovorum, B. halodurans, and/or so forth);and/or those described herein. The transpososome HAM constructs may beintroduced into the organism by any technique known in the art, such as,but not limited to, electroporation, and so forth. Such techniques areknown in the art and are detailed in Sambrook J. C., E. F. Fritsch, andT. Maniatas, 1989. Molecular Cloning: A Laboratory Manual, 2nd ed., ColdSpring Harbor Laboratory Press, N.Y, p. 6.3-6.34; or Sambrook andRussell, Molecular Cloning: A Laboratory Manual (3-Volume Set), 3^(rd)ed. (available online at molecularcloning.com/); both referencesincorporated by reference herein.

Example 2(b): Recombination/Integration of Hydrolysis AssociatedMolecules into Genome of Organism Able to Ferment Sugar to Ethanol

One or more HAMs are integrated into the genome of an organism that isable to ferment and/or convert sugar molecules to a product. Atranspososome is made that includes the HAMs and contains sequences forpromoting the integration of the included HAM(s) into the genome. Thetranspososome construct may be any type and/or kind of transpososomeconstruct known in the art, such that the transpososome constructassists in the integration of the one or more HAMs into a host genome.Such transpososome may include Mu based transpososomes or the like.

Example 3: Transformation/Expression of Hydrolysis Associated Moleculesand Fermentation Associated Molecules in an Organism

Both HAMs and FAMs are expressed in a suitable organism. The HAMs andFAMs to be expressed include any and/or all of the Hydrolysis orFermentation Associated Molecules as described herein.

Example 3(a): Transformation of Hydrolysis Associated Molecules andFermentation Associated Molecules Using a Transpososome

One or more transpososomes are constructed including HAMs and/or FAMs.Similar to Example 1 and Example 2, included in each of thetranspososomes are the nucleic acids necessary for insertion andexpression of the nucleic acids in the transpososome. Included in thetranspososome are the nucleic acids necessary for insertion of thetransposon, copies of the MuA transposase, Mu transposon ends, andprovision for expression of the nucleic acids contained in thetranspososome in the particular organism in which they are to beutilized. Transpososomes may also be constructed using sequences andelements optimized for use in gram-positive bacteria. See, Pajunen etal., Microbiology (2005) 151, 1209-1218. Conventional techniques may beused to construct the transpososome. Additionally, the constructionand/or insertion of desired nucleic acids into transpososomes is wellknown in the art, see, Pajunen et al. It is contemplated that multipletranspososomes may be constructed with the HAMs and FAMs, and mayinclude a variety of promoters and/or enhancers that may function toenhance expression of HAMs and/or FAMs.

Once constructed, the transpososomes are introduced into a targetorganism. Some non-limiting examples of potential organisms include E.coli, any type and/or kind of thermophilic bacteria (e.g., Sulfolobus,Thermoproteus, etc.), and/or so forth. The transpososome constructs maybe introduced into the organism by any technique known in the art, or asdescribed herein.

Example 3(b): Integration of Hydrolysis and Fermentation AssociatedMolecules into Genome of Organisms

One or more FAMs and/or HAMs are integrated into the genome of A.acidocaldarius. A transpososome is made that includes FAMs and/or HAMsand contains sequences for promoting the integration of the includedFAMs and/or HAMs into the genome. The transpososome may be any typeand/or kind of transpososome construct known in the art, such that thetranspososome assists in the integration of the one or more FAMs and/orHAMs into a host genome. Such transpososomes may include Mu basedtranspososomes or the like.

All references, including publications, patents, and patentapplications, cited herein are hereby incorporated by reference to thesame extent as if each reference were individually and specificallyindicated to be incorporated by reference and were set forth in itsentirety herein.

While this invention has been described in certain embodiments, thepresent invention can be further modified within the spirit and scope ofthis disclosure. This application is therefore intended to cover anyvariations, uses, or adaptations of the invention using its generalprinciples. Further, this application is intended to cover suchdepartures from the present disclosure as come within known or customarypractice in the art to which this invention pertains and which fallwithin the limits of the appended claims and their legal equivalents.

Example 4 RAAC02661: A Xylan Alpha-Glucuronidase

Provided in SEQ ID NO:336 is a nucleotide sequence isolated fromAlicyclobacillus acidocaldarius and encoding the polypeptide of SEQ IDNO:337. As can be seen in FIGS. 21A-21D, SEQ ID NO:337 aligns well withother proteins identified as xylan alpha-glucuronidases. Of particularimportance, it is noted that where amino acids are conserved in otherxylan alpha-glucuronidases, those amino acids are generally conserved inSEQ ID NO:337. Thus, the polypeptide provided in SEQ ID NO:337 isproperly classified as a xylan alpha-glucuronidase.

The polypeptides of SEQ ID NOs:348-352 are representative examples ofconservative substitutions in the polypeptide of SEQ ID NO:337 and areencoded by the nucleotide sequences of SEQ ID NOs:343-347, respectively.

The nucleotide sequences of SEQ ID NOs:336 and 343-347 are placed intoexpression vectors using techniques standard in the art. The vectors arethen provided to cells such as bacteria cells or eukaryotic cells suchas Sf9 cells or CHO cells. In conjunction with the normal machinerypresent in the cells, the vectors comprising SEQ ID NOs:336 and 343-347produce the polypeptides of SEQ ID NOs:337 and 348-352. The polypeptidesof SEQ ID NOs:337 and 348-352 are then isolated and/or purified. Theisolated and/or purified polypeptides of SEQ ID NOs:337 and 348-352 arethen demonstrated to have activity as xylan alpha-glucuronidases.

The isolated and/or purified polypeptides of SEQ ID NOs:337 and 348-352are challenged with polysaccharides, lignocellulose, cellulose,hemicellulose, lignin, starch, chitin, polyhydroxybutyrate,heteroxylans, glycosides, xylan-, glucan-, galactan-, and/ormannan-decorating groups. The isolated and/or purified polypeptides ofSEQ ID NOs:337 and 348-352 are demonstrated to have activity in at leastpartially degrading, cleaving, and/or removing polysaccharides,lignocellulose, cellulose, hemicellulose, lignin, starch, chitin,polyhydroxybutyrate, heteroxylans, glycosides, xylan-, glucan-,galactan-, and/or mannan-decorating groups.

Example 4(a) Production and Purification of RAAC02661

The nucleotide sequence of SEQ ID NO:337 was cloned fromAlicyclobacillus acidocaldarius. SEQ ID NO:336 encodes the polypeptideof SEQ ID NO:337. SEQ ID NO:336 was cloned into the pBAD/HIS Aexpression vector for E. coli and provided to E. coli viaelectroporation. Expression of SEQ ID NO:337 was detected fromtransformed E. coli comprising SEQ ID NO:336 and RAAC02661 was affinitypurified using a cobalt resin for activity testing.

Example 4(b) α-glucuronidase (AGUR) Activity of RAAC02661

RAAC02661 purified from E. coli was tested for XYL activity using anassay summarized as follows:

A solution of aldouronic acids (AUAs) was created by diluting 50 μL of amixture of aldotetraouronic acid, aldotriouronic acid and aldobiouronicacid (40:40:20; Aldouronic Acid Mixture, Megazyme Cat. No. O-AMX) with1.95 mL of an appropriate buffer at 50 mM for pHs ranging from 1 to 9.Buffers included maleic acid (pH 1.0-2.0), Glycine HCl (pH 3.0), sodiumacetate (pH 3.5-5.0), sodium phosphate (pH 6.0-8.0), Tris-HCl (pH 9.0),and CAPS buffer (pH 10.0).

Samples of purified RAAC02661 generated in Example 38 were diluted to anappropriate concentration for activity measurement in the appropriatebuffer at 50 mM for pHs ranging from 1 to 10. Samples (RAAC02661 samplesand positive controls) were placed in the wells of a 96-well plate in 10μL aliquots. Blanks of buffer only were placed in some wells. AUAsolution, preheated to 50, 60, 70, 80, or 90 degrees Celsius, was thenadded to each well and the plate was incubated at 50, 60, 70, 80, or 90degrees Celsius for 3 minutes. Dinitrosalicylic acid solution was thenadded to each well and the plate was further incubated at 80 degreesCelsius for an additional 10 minutes. The AGUR activity was measuredusing a 96-well plate reader (Molecular Devices UV-Vis) at a wavelengthof 540 nm. Specific activity for RAAC02661 as determined appears in FIG.2.

BIBLIOGRAPHIC REFERENCES

-   Garrote, G., H. Dominguez, and J. C. Parajo, 2001, Manufacture of    xylose-based fermentation media from corncobs by posthydrolysis of    autohydrolysis liquors, Appl. Biochem. Biotechnol., 95:195-207.-   Hamelinck, C. N., G. van Hooijdonk, and A. P. C. Faaij, 2005,    Ethanol from lignocellulosic biomass: techno-economic performance in    short-, middle-, and long-term, Biomass Bioenergy, 28:384-410.-   Laitinen et al., 2005, A Multipurpose Vector system for the    screening of libraries in bacteria, insect and mammalian cells and    expression in vivo, Nucleic Acids Research 33(4).-   Liu C., and C. E. Wyman, 2003, The effect of flow rate of compressed    hot water on xylan, lignin, and total mass removal from corn stover,    Ind. Eng. Chem. Res., 42:5409-5416.-   Lynd et al., 2002, Micro. and Mol. Biol. Rev., vol. 66, No. 3, pp.    506-577.-   Sambrook J. C., E. F. Fritsch, and T. Maniatas, 1989. Molecular    Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory    Press, N. Y, pp. 6.3-6.34.-   Sambrook and Russell, 2001, Molecular Cloning: A Laboratory Manual    (3-Volume Set), 3^(rd) ed. (available online at    molecularcloning.com/).-   Ng et al., 1981, Applied and Environmental Microbiology, 41(6):    1337-1343.-   Tsao, G. T., M. R. Ladisch, and H. R. Bungay, 1987. Biomass    Refining, In Advanced Biochemical Engineering, Wiley Interscience,    N.Y., pp. 79-101.

What is claimed is:
 1. A genetically modified bacteria or funguscomprising: at least one exogenous nucleic acid encoding a polypeptidehaving at least 90% sequence identity to SEQ ID NO: 304; and wherein thepolypeptide has carboxylesterase type B activity.
 2. The geneticallymodified bacteria or fungus of claim 1, wherein the at least oneexogenous nucleic acid is integrated into the host organism's genome. 3.The genetically modified bacteria or fungus of claim 1, wherein thepolypeptide has carboxylesterase type B activity at or above fiftydegrees Celsius.
 4. The genetically modified bacteria or fungus of claim1, wherein the polypeptide comprises SEQ ID NO:
 304. 5. The geneticallymodified bacteria or fungus of claim 1, wherein the at least oneexogenous nucleic acid has at least 90% homology to SEQ ID NO:303. 6.The genetically modified bacteria or fungus of claim 1, wherein the atleast one exogenous nucleic acid is SEQ ID NO:303.
 7. An extractisolated from the genetically modified bacteria or fungus of claim 1,wherein the extract comprises the polypeptide having carboxylesterasetype B activity and a polypeptide natively produced by the geneticallymodified organism.
 8. A method for at least partially processinghemicellulose into a product, the method comprising: contacting a liquidculture medium of the genetically modified bacteria or fungus of claim 1with hemicellulose, wherein the liquid culture medium comprises thepolypeptide having carboxylesterase type B activity.
 9. The methodaccording to claim 8, wherein the contacting occurs at or above fiftydegrees Celsius.
 10. A method for at least partially processinghemicellulose, the method comprising: isolating an enzyme extract fromthe genetically modified bacteria or fungus of claim 1; and contactingthe extract with hemicellulose, wherein the extract comprises thepolypeptide having carboxylesterase type B activity and a polypeptidenatively produced by the genetically modified organism.
 11. A method ofproducing a product from biomass, the method comprising: pretreating thebiomass with an acid; enzymatically hydrolyzing cellulose andhemicellulose in the biomass to produce monomeric sugars andconcurrently fermenting the monomeric sugars to a product; andrecovering the product, wherein at least a portion of the hydrolyzing ofcellulose and hemicellulose or the fermentation is performed by thegenetically modified bacteria or fungus of claim
 1. 12. A method ofproducing a product from biomass, the method comprising: pretreating thebiomass with an acid; enzymatically hydrolyzing cellulose andhemicellulose in the biomass to produce monomeric sugars; fermenting themonomeric sugars to a product; and recovering the product, wherein atleast a portion of the hydrolyzing of cellulose and hemicellulose or thefermentation is performed by the genetically modified bacteria or fungusaccording to claim 1, and wherein the pretreatment, the hydrolyzing ofcellulose and hemicellulose, and the fermentation are performedconcurrently.
 13. A method of processing biomass, the method comprising:treating the biomass with an acid and concurrently enzymaticallyhydrolyzing cellulose and hemicellulose in the biomass to producemonomeric sugars, wherein at least a portion of the hydrolyzing ofcellulose and hemicellulose is performed by the genetically modifiedbacteria or fungus of claim
 1. 14. The genetically modified bacteria orfungus of claim 1, wherein the exogenous nucleic acid is contained in avector.